Nigella sativa seed extracts and its oil have been exploited for their various health benefits. In this study, the effects of N. sativa oil on tramadol-induced tolerance and dependence and possible mechanism(s) of these effects were investigated, for the first time, in mice. Repeated administration of N. sativa oil (4 ml/kg, p.o.) inhibited the development of tramadol tolerance, as measured by the hot plate test, and dependence as assessed by naloxone (5mg/kg, i.p.)-precipitated withdrawal manifestations. Concomitantly, nitric oxide overproduction and increase in brain malondialdehyde level induced by repeated administration of tramadol to mice or by administration of naloxone to tramadol-dependent mice were inhibited by co-administration of the oil. Also, the decrease in brain intracellular reduced glutathione level and glutathione peroxidase activity induced by both treatments was inhibited by co-administration of the oil. The increase in brain glutamate level induced by both treatments was not inhibited by concurrent administration of the oil. The inhibitory effect of N. sativa oil on tramadol-induced tolerance and dependence was enhanced by concurrent i.p. administration of the NMDA receptor antagonist, dizocilpine (0.25mg/kg). Also, the inhibitory effect of the oil on naloxone-induced biochemical alterations in tramadol-dependent mice was enhanced by concurrent administration of dizocilpine. Similarly, concurrent i.p. administration of the NO synthase inhibitor, L-N(G)-nitroarginine methyl ester (10mg/kg) or the antioxidant, N-acetylcysteine (50mg/kg) enhanced these inhibitory effects of N. sativa oil. On the other hand, these effects were antagonized by concurrent i.p. administration of the NO precursor, L-arginine (300 mg/kg). These results provide evidence that N. sativa oil appears to have a therapeutic potential in tramadol tolerance and dependence through blockade of NO overproduction and oxidative stress induced by the drug.